Positional cloning by fast-track SNP-mapping in Drosophila melanogaster.
Positional cloning of chemically induced mutations is the rate-limiting step in forward genetic screens in Drosophila. Single-nucleotide polymorphisms (SNPs) are useful markers to locate a mutated region in the genome. Here, we provide a protocol for high-throughput, high-resolution SNP mapping that enables rapid and cost-effective positional cloning in Drosophila. In stage 1 of the protocol, we use highly multiplexed tag-array mini-sequencing assays to map mutations to an interval of 1-2 Mb. In these assays, SNPs are genotyped by primer extension using fluorescently labeled dideoxy-nucleotides. Fluorescent primers are captured and detected on a microarray. In stage 2, we selectively isolate recombinants within the identified 1-2 Mb interval for fine mapping of mutations to about 50 kb. We have previously demonstrated the applicability of this protocol by mapping 14 muscle morphogenesis mutants within 4 months, which represents a significant acceleration compared with other commonly used mapping strategies that may take years.
Schnorrer
Frank
F
Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria. schnorrer@biochem.mpg.de
Ahlford
Annika
A
Chen
Doris
D
Milani
Lili
L
Syvänen
Ann-Christine
AC
D000818:Animals; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D004625:Embryo, Nonmammalian; D005819:Genetic Markers; D005838:Genotype; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011995:Recombination, Genetic; D012984:Software
D005819:Genetic Markers